DESCRIPTION (Verbatim From the Applicant's Abstract): The major aim of this application is to understand the molecular mechanisms that control growth cone guidance. The research funded by this application continues to focus on guidance at a single choice point: the midline. Most growth cones in the CNS confront the midline and make a series of specific guidance decisions. They grow towards or away from the midline. Once near the midline, they decide to cross or not to cross the midline. For those that do cross the midline, they decided to never cross it again. Genetic analysis has led to the discovery of many of the key ligands and receptors controlling these decisions. The ongoing success of this genetic approach is based on two premises: (i) the power of the genetics as a discovery tool for identifying gene function; and (ii) the remarkable evolutionary conservation of gene structure and function across species. In the studies described here, genetic analysis is used in the model system: the fruit fly Drosophila. Genes controlling midline guidance are first identified in Drosophila; subsequent identification of the their mammalian homologues is facilitated using rapidly-advancing genomics capabilities. The mammalian homologues of these guidance genes appear to be expressed and function in a similar fashion, and are likely to control important aspects of human brain and spinal cord development. Thus, the genes discovered using genetic analysis in this model system may hold the key for new insights into spinal cord regeneration and repair of nervous system injury. Midline cells secrete Netrins and Slits. Growth cones express differential levels of receptors for both ligands: Frazzled (the DCC homologue), an attractive Netrin receptor, and Robo, a repulsive Slit receptor. A second Slit receptor -- Robo2-- has been discovered and will be analyzed. A membrane protein, commissureless, regulates the level of Robo. A second Comm-like protein, Comm2, has been discovered. This application is aimed at understanding how these ligands, receptors, and regulators control midline guidance. Questions will be answered about how the levels and signaling of these ligands and receptors control these guidance decisions, how these receptors transduce signals that control steering, how growth cones make dynamic changes in receptors as they navigate this choice point, and how growth cones make cell-specific decisions. A multidisciplinary approach will be used to understand how guidance works at this choice point. The goal of this research is to understand how differences in binding, signaling, and regulation of the Robo and Robo2 receptors, their ligand Slit, and their regulators Comm and Comm2, help control the well defined but complex set of guidance decisions at the midline.